Cyclic nucleotide-gated channel block by hydrolysis-resistant tetracaine derivatives

Article abstract of DOI:10.1021/jm200495g

To meet a pressing need for better cyclic nucleotide-gated (CNG) channel antagonists, we have increased the biological stability of tetracaine-based blockers by synthesizing amide and thioamide linkage substitutions of tetracaine (1) and a higher affinity

Full text of DOI:10.1021/jm200495g

ARTICLE
pubs.acs.org/jmc
Cyclic Nucleotide-Gated Channel Block by Hydrolysis-Resistant
Tetracaine Derivatives
Adriana L. Andrade,† Kenneth Melich,† G. Gregory Whatley,‡ Sarah R. Kirk,‡ and Jeffrey W. Karpen*,†
†Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University,
3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States
‡Department of Chemistry, Willamette University, Salem, Oregon 97301, United States
ABSTRACT: To meet a pressing need for better cyclic nucleotide-gated (CNG) channel
antagonists, we have increased the biological stability of tetracaine-based blockers by synthesizing
amide and thioamide linkage substitutions of tetracaine (1) and a higher affinity octyl tail derivative
(5). We report the apparent KD values, the mechanism of block, and the in vitro hydrolysis rates for
these compounds. The ester linkage substitutions did not adversely affect CNG channel block;
unexpectedly, thioamide substitution in 1 (compound 8) improved block significantly. Further-
more, the ester linkage substitutions did not appear to affect the mechanism of block in terms of the strong state preference for closed
channels. All ester substituted compounds, especially the thioamide substitutions, were more resistant to hydrolysis by serum
cholinesterase than their ester counterparts. These findings have implications for dissecting the physiological roles of CNG channels,
treating certain forms of retinal degeneration, and possibly the current clinical uses of compound 1.
’ INTRODUCTION
channels leads to the conclusion that much research remains to
be done on better CNG channel antagonists.
Cyclic nucleotide-gated (CNG) ion channels are known
for their role in phototransduction in retinal photoreceptors
and in odorant transduction in the olfactory epithelium.1,2
CNG channels are also present in other brain regions and
nonsensory tissues, but their physiological roles are much
less clear.3ꢀ7 CNG channel activation in photoreceptors is
regulated by the cytoplasmic concentration of cGMP, which
binds to and opens the channel to allow influx of Naþ and
Ca2þ ions. Alterations of CNG channel activity have been
observed in some forms of retinitis pigmentosa, a group of
inherited diseases that cause progressive degeneration of rod
and cone photoreceptors.8ꢀ14 Mutations that cause elevated
In recent years, our group has developed a number of high
affinity CNG channel blockers using 1 as a scaffold.38ꢀ40
Compound 1 is clinically approved for temporary anesthesia
in various surgical procedures, including those involving the
eye.41,42 Its effects are localized and short-lived because of its
rapid degradation by esterases.43 One major improvement in
the design of a tetracaine-based CNG channel blocker would
be to increase its biological stability against hydrolysis. In this
study, we have synthesized amide and thioamide linkage
versions of 1, as well as its relative 5 that has a higher affinity
for CNG channels.40 We report the apparent KD values and in
vitro hydrolysis rates by serum cholinesterase (butyryl-
cholinesterase) for these novel compounds. We believe we
have uncovered promising compounds not only for CNG
channel research but for the treatment of specific forms of
retinal degeneration as well. Furthermore, these improve-
ments may have implications for current clinical usage of 1
in general.
cGMP levels lead to prolonged channel activation and Ca2þ
-
triggered cell death.10,12,14ꢀ17 In mouse models, reduction of
CNG channel activity strongly correlated with improve-
ments in the overall progression of the disease18ꢀ20 (see
also refs 21 and 22), but unfortunately, there are no clinically
approved drugs that target CNG channels.
Compared to voltage-gated channels, CNG channel pharma-
cology is quite unsophisticated.20,23 The most widely used CNG
channel antagonist in research, l-cis-diltiazem, is an incomplete
blocker and relatively nonspecific.24ꢀ27 CNG channels are also
blocked by some local anesthetics, one example being tetracaine
[2-(dimethylamino)ethyl 4-(butylamino)benzoate] (1).28ꢀ30
Compound 1 blocks CNG channels with relatively high affinity,
although differently from voltage-gated sodium channels. Like
sodium channels, the interaction of 1 with CNG channels is
thought to be in the selectivity filter and the pore region.31ꢀ35
However, 1 has a greater affinity for the sodium channel in its
open, inactivated-state,36 whereas for CNG channels, it binds
with higher affinity to the closed state.37 The inability of 1 and
l-cis-diltiazem to distinguish between CNG channels and other
’ CHEMISTRY
Compound 1 derivatives were prepared according to Scheme 1.
An alkyl substituent was added to the amino end of 4-aminoben-
zoic acid (2) via reductive amination using a synthesis adapted
from Sato et al.44 The resulting alkylated benzoic acid derivatives
(3 and 4) were then activated at the carboxylic acid with 1,
10-carbonyldiimidazole (CDI) and subsequently esterified or
amidated using 2-(dimethylamino)ethanol or N0,N0-dimethy-
lethane-1,2-diamine, respectively, to yield target compounds
Received: April 21, 2011
Published: June 02, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Tetracaine Derivativesa
a Reagents and conditions: (i) butanal or octanal, R-picoline-borane, MeOH, room temp, 16ꢀ24 h (88%, 94%); (ii) CDI, DME, 60 °C, 2 h, followed by
2-(dimethylamino)ethanol, NaH, 60 °C to room temp, 16ꢀ24 h (95%); (iii) CDI, DME, 60 °C, 2 h, followed by N0,N0-dimethylethane-1,2-diamine,
NaH, 60 °C to room temp, 16ꢀ24 h (93%, 87%); (iv) Lawesson’s reagent, toluene, reflux, 2.5 h (26%, 73%).
5ꢀ7.45 Compounds 6 and 7 were further treated with Lawesson’s
reagent to yield target thioamide compounds 8 and 9.46
A small transient decay in current attributed to an ion
accumulation effect was seen with each voltage step with large
currents (typically >1 nA, Figures 1 and 2).48 Corrections for ion
accumulation did not substantially change previous KD estimates
for 1 (6.8 μM corrected for ion accumulation39 and 6.7 μM not
corrected,40 both determined at þ40 mV). We found that the KD
value estimates taking this effect into account did not change the
relationship among the compounds for block; hence, it was not
corrected for in the values reported here.
’ RESULTS
CNG Channel Block at Saturating cGMP. The effectiveness
of retinal rod CNG channel current block by the ester linkage-
substituted tetracaine derivatives was tested in Xenopus oocyte
preparations. Excised, inside-out patches pulled from oocytes
expressed heteromeric rod CNG channels consisting of CNGA1
and CNGB1 subunits. This was verified by substantial block of
2 mM cGMP-induced currents with 20 μM l-cis-diltiazem (74.2
( 5.8% at Vm = þ50 mV).47
The KD values at both þ50 and ꢀ50 mV for all compounds
tested are plotted in Figure 3. The mean KD values are summarized
in Table 1, along with estimated logP values for each compound.
On the basis of these results, it is apparent that the headgroup
linkage of 5 has a limited role in CNG channel current block.
However, substitutions of the headgroup linkage in 1, in particular
the carbonyl oxygen, play a direct role inCNG channel interaction.
While the amide substitution of the ester linkage of 1 (compound
6) has little effect on the KD values, the thioamide substitution
unexpectedly improves the effectiveness of block (compound 8).
State Dependence of Block. Compound 1 has been reported
to preferentially block CNG channels in the closed conforma-
tion, and the effectiveness of block improves with half-maximal
channel activation.37 Although we previously established that 5
has a greater affinity for CNG channels under saturating con-
centrations of cGMP than 1, we did not examine if the mechan-
ism of block was different. We sought to determine if the ester
linkage-substituted compounds, as well as compound 5, exhibit
state preference for block. We measured the apparent KD values
at both positive and negative potentials for CNG channel
currents activated by saturating (2 mM) and subsaturating
concentrations of cGMP (50 and 100 μM). Under these condi-
tions, with no exceptions, the determined KD values for all
compounds were smaller at subsaturating cGMP than at saturat-
ing cGMP. The KD values at subsaturating cGMP (at þ50 mV)
normalized to the KD values at saturating cGMP are plotted
against 1 ꢀ I/Imax, which is related to the fraction of closed
channels, for compounds 1, 8, 5, and 9 in Figure 4. The solid lines
are simulations for the expected relationship between KD and
fraction of closed channels for an exclusive closed channel
blocker, using three previously determined estimates for the
Each compound’s apparent affinity for the heteromeric CNG
channel was determined under maximal channel activation
(2 mM cGMP). CNG channel currents were elicited by a voltage
step protocol to ꢀ50 and þ50 mV (Figures 1A and 2A, insets).
The apparent KD value at each membrane potential was esti-
mated first by determining IþB and IꢀB at steady state, where I
þB
is the current in the presence of blocker and IꢀB is current in the
absence of blocker, for different blocker concentrations ([B]).
The following equation for block at a single binding site was fit to
the data to obtain KD:
I
¼ IꢀBKD=ðKD þ ½BꢁÞ
þB
Representative traces for CNG channel currents at positive and
negative membrane potentials activated by 2 mM cGMP, as well
as graphs with fitted curves, are shown for compounds 1, 8, 5, and
9 in Figures 1 and 2. As reported previously,40 5 had a higher
affinity for CNG channels than 1 (Figures 1 and 2). Compound 8
proved to be a higher affinity CNG channel blocker compared to
compound 1 at both positive and negative membrane potentials
(Figure 1). In contrast, compound 9 with the same headgroup
linkage as compound 8 had a similar CNG channel affinity
compared to compound 5 (Figure 2). From Figures 1A and 2A, it
is also possible to note the characteristic voltage dependence of
block of compounds 1 and 5, which improves with positive
membrane potentials. All compounds tested exhibited voltage-
dependent block, although the voltage-dependence of 5 and its
derivatives was appreciably less than that of 1 and its derivatives.
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Figure 1. Heteromeric rod CNG channel block by compounds 1 and 8.
Leak-subtracted currents from a representative excised inside-out patch
from oocytes expressing heteromeric rod CNG channels (A). Currents
were elicited by a voltage step protocol from 0 to ꢀ50 to þ50 mV (inset)
in the presence of 2 mM cGMP or 2 mM cGMP and compound (5 μM).
Time scale is shown in the lower left-hand corner of the panel and inset,
and the zero current level is indicated by the dotted line. (B) Currents
obtained from a concentration series of compounds 1 (white) and 8
(black) are plotted against compound concentration. Solid line indicates
the fit of the equation for block at a single binding site (see text). KD
values determined from the fit of the equation were 4.2 μM at þ50 mV
and 15.6 μM at ꢀ50 mV for compound 1, and 0.5 μM at þ50 mV and
3.7 μM at ꢀ50 mV for compound 8.
Figure 2. Heteromeric rod CNG channel block by compounds 5 and 9.
Leak-subtracted currents from a representative excised inside-out patch
from oocytes expressing heteromeric rod CNG channels (A). Currents
were elicited by a voltage step protocol from 0 to ꢀ50 to þ50 mV (inset)
in the presence of 2 mM cGMP or 2 mM cGMP and compound (5 μM).
Time scale is shown in the lower left-hand corner of the panel and inset,
and the zero current level is indicated by the dotted line. (B) Currents
obtained from a concentration series of compounds 5 (white) and 9
(black) are plotted against compound concentration. Solid line indicates
the fit of the equation for block at a single binding site (see text). KD
values determined from the fit of the equation were 1.5 μM at þ50 mV
and 1.8 μM at ꢀ50 mV for compound 5, and 1.3 μM at þ50 mV and 1.8
μM at ꢀ50 mV for compound 9.
with an expected closed channel blocker. Thus, increasing the
butyl tail of compound 1 to eight carbons (compound 5), as well
as substituting the ester linkages of both 1 and 5, does not appear
to alter the essential mechanism of CNG channel block.
Resistance to in Vitro Hydrolysis. In addition to the unexpected
increase in apparent affinity for CNG channels by compound 8, the
probability of closed heteromeric retinal rod channels at saturat-
ing cGMP (aꢀc).49ꢀ51 The dotted line is a simulation for a
blocker with no preference for state, while the dashed line is a
simulation for an exclusive open channel blocker. Despite
inherent variability in the relationships between KD values and
fractional current, the data for all compounds track much better
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Table 1. KD Value Determinations, Estimated Log P, and in Vitro Serum Cholinesterase Hydrolysis Rates for Tetracaine (1) and
Derivatives
a The compounds are shown here in what is expected to be the predominant protonation state at pH 7.6. b Calculated for the unprotonated forms using
ALOGPS 2.1 (Virtual Computational Chemistry Laboratory). c Not detected.
amide and thioamide linkage substitutions should provide an
improvement for many in vivo applications or tissue preparations
to a tetracaine-based CNG channel blocker in terms of biological
stability. Compound 1 is rapidly hydrolyzed by butyrylcholinester-
ase in the bloodstream,43 hence, its limitation to local targets in
clinical usage. Butyrylcholinesterase is also the predominant esterase
present in ocular tissues.52ꢀ54 Similarly, in vivo use of a tetracaine-
based CNG channel blocker would be short-lived. Amide and
thioamide linkages are more resistant to hydrolysis than ester
linkages. We therefore tested the resistance of the ester linkage-
substituted compounds to in vitro hydrolysis using butyrylcholines-
terase purified from human blood serum (Table 1). The concentra-
tions of compounds used in the assays were well in excess of
butyrylcholinesterase’s Km for 1 and other local anesthetics.43 The
amide linkage substitutions of compounds 6 and 7 increased the
resistance to hydrolysis substantially. The increase in hydrolysis
resistance was even more dramatic for the thioamide linkage
substitutions (compounds 8 and 9), which proved to be much
too stable for our system to detect any hydrolysis product, even after
24 h incubations.
rates, for four novel tetracaine-based compounds with potentially
increased biological stabilities (compounds 6, 7, 8, and 9). The
KD values obtained for 1 and 5 (Table 1) were similar to those
previously reported.39,40 As expected, 5 had a higher apparent
affinity for CNG channels than 1. The substituted amide linkages
(compounds 6 and 7) did not adversely affect the apparent
affinity for CNG channel block (Table 1). In addition, these
compounds were more resistant to esterase hydrolysis than their
ester counterparts (compounds 1 and 5). This poses a significant
advantage for their use in in vivo research or in the clinic.
Compounds 8 and 9 were even more resistant to esterase
hydrolysis than compounds 6 and 7, attributed to the thioamide
linkage substitution (Table 1). For compound 9 the thioamide
substitution did not enhance the apparent affinity for CNG
channels, but it did for compound 8. It is unknown why the
thioamide substitution in compound 8 improved CNG channel
block, as the precise structure of the channel pore remains
unresolved. These results suggest that 1 and its derivatives bind
at a different site in the channel than 5 and its derivatives. The
observation is further supported by the exhibited differences in
voltage dependence of block. It is possible that the lower
electronegativity or increased size of sulfur in lieu of the carbonyl
oxygen enhances the interaction of compound 8, improving the
apparent affinity for the channel pore. It is also possible that the
presence of sulfur instead of oxygen increases the partial double
bond character of the carbonꢀnitrogen bond, affecting the
orientation of the aromatic and headgroup moieties.
One concern relating to the amide linkage (compounds 6 and 7)
is that it could increase the susceptibility of hydrolysis by proteases
present in the eye.55,56 However, this was not the case. Using the
broad-spectrum, nonspecific endopeptidases chymotrypsin and
proteinase K, we were not able to detect any hydrolysis products
for all compounds, while hydrolysis products of 4-nitrophenyl
acetate and BSA were detected as positive controls.
Compound 1 has been reported to be a closed-channel
blocker.37 Although we have previously determined the KD
values for 5, its mechanism of block had not yet been examined.
Compound 5, as well as all compounds in this study, exhibited
preference for the closed state over the open state (Figure 4).
’ DISCUSSION
We have synthesized and determined the apparent affinities
and mechanism of CNG channel block, as well as the hydrolysis
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greater stability of the amide and thioamide linkages should
enhance the half-life. Particularly for compounds 8 and 9, the
clearance rate would likely depend on other metabolic pathways,
but this would need to be determined in a proper pharmacoki-
netic study. One question that emerges from this study is which
compound appears to have the greatest potential for clinical use?
We are contemplating only local tissue uses of these compounds.
Nonetheless, the stability of compounds 8 and 9 against hydro-
lysis is so dramatic that their safety for clinical use might come
into question. The high stability of the thioamide linkage in a
tetracaine-based CNG channel blocker may increase the chances
for toxic systemic reactions. Large systemic doses of thiobenza-
mide and its para-substituted derivatives have been shown to be
hepato- and nephrotoxic,60ꢀ62 and certain thioamides such as
propylthiouracil inhibit thyroid peroxidase and are used to treat
hyperthyroidism.63 These issues remain to be examined at the
low doses needed to achieve CNG channel block. Lastly,
compounds 7 and 9 have larger estimated log P values than
compounds 6 and 8 (Table 1) and as such would theoretically
have better accessibility to reach channel targets.
This study may have broader implications than for CNG
channel blockers alone. There are situations in which longer
lasting anesthetics are desirable, and there have been several
attempts to achieve this.64ꢀ66 The amino amide type of local
anesthetics are well recognized for their better biological stability
than amino esters, but the thioamide linkage, to our knowledge,
is the first of its kind. Indeed, there have been some attempts to
increase the half-life of 1 in biological preparations.67ꢀ70 The
hydrolysis resistant versions of 1 reported here may be attractive
options for longer lasting local anesthetics; however, their effects
on nerve conductance need to be further examined.
Figure 3. KD values determined from all experimental patches. Plots
show all KD values determined at þ50 mV (upper panel) and ꢀ50 mV
(lower panel). Solid horizontal brackets with asterisks indicate groups
significantly different from compound 1 using the HolmꢀSidak method
for multiple pairwise comparisons; P < 0.01.
’ EXPERIMENTAL SECTION
Retinal Rod CNG Channel Expression in Oocytes. Ovaries
were surgically removed from adult Xenopus laevis females (Xenopus
Express; Brooksville, FL) anesthetized with ice-cold 0.1% tricaine and
0.1% NaHCO3 solution. Oocytes were chemically released from ovarian
follicles in Ca2þ-free Barth’s solution (in mM: 88 NaCl, 1 KCl, 2.4
NaHCO3, 0.82 MgSO4, 7.5 Tris, 2.5 sodium pyruvate, 100 U/mL
penicillin, and 100 μg/mL streptomycin, pH 7.4) containing 0.1 U/mL
Liberase Blendzymes (Roche, Indianapolis, IN). Stages IV and V
oocytes were visually sorted and stored in ND-96 (in mM: 96 NaCl, 2
KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, 2.5 sodium pyruvate, 100 U/mL
penicillin, and 100 μg/mL streptomycin, pH 7.4) at 16 °C. Oocytes were
co-injected the following day with 33 ng of CNGA1 and 67 ng of
CNGB1 cRNA (2:1) synthesized from linearized pGEM-HE expression
vectors containing the channel subunit cDNA sequences71 using
T7 mMESSAGE mMACHINE (Ambion, Austin, TX). Injected oocytes
were incubated at 18 °C the first day and 16 °C for the remaining days.
Electrophysiological Recordings. Recordings from inside-out
excised patches were made 3ꢀ7 days after oocyte injection on an
Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Briefly,
oocyte vitelline membranes were removed in solution containing
(in mM) 200 K aspartate, 20 KCl, 1 MgCl2, 10 EGTA, 10 HEPES,
pH 7.4. Oocytes were placed in a recording chamber in solution
containing (in mM) 130 NaCl, 2 HEPES, 0.02 EDTA, 1 EGTA, pH
7.6, and borosilicate glass electrodes (1ꢀ3 MΩ) were filled with
identical solution. Macroscopic currents were filtered at 1 kHz and
sampled at 2 kHz using pCLAMP 8.0 software (Axon Instruments).
Channels were either fully activated with 2 mM or partially activated
with 50 and 100 μM cGMP. Solutions containing different tetracaine
derivatives were exchanged using a RSC-100 rapid solution changer
The physiological concentration of free cGMP in dark-adapted
rod outer segments has been estimated to be about 3.5 μM.57,58
Given the very large fraction of closed channels, the potency for
CNG channel block in the intact retina is likely to be even greater
than the values reported here.
It was a concern that the amide bond in compounds 6 and 7
could be susceptible to hydrolysis by proteases; however, we
found that they were resistant to both proteinase K and
chymotrypsin. Chymotrypsin preferentially catalyzes the hydro-
lysis of peptide bonds involving L-isomers of tyrosine, phenyla-
lanine, and tryptophan on the carboxy side.59 As the amide bond
adjacent to the aromatic ring in compounds 6 and 7 resembles a
peptide bond of an aromatic amino acid residue, it was plausible
that it would be recognized by the hydrophobic pocket of the
chymotrypsin catalytic site. The resistance of compounds 6 and 7
to chymotrypsin may be due to the lack of a “carbon backbone” in
the structure. A similar argument can be applied to explain the
resistance to hydrolysis by proteinase K: the aromatic ring
directly adjacent to the equivalent of a peptide bond in com-
pounds 6 and 7 renders it unrecognizable by the endopeptidase.
Not surprisingly, compounds 8 and 9 with thioamide linkages
were also completely resistant to protease action.
Compound 1’s actions are very short-lived, being rapidly
degraded mainly by butyrylcholinesterase in the blood. The
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Figure 4. State dependence of block. Plots showing relationship of all KD values determined for compounds 1 (2 μM), 8 (2 μM), 5 (1 μM), and 9 (1 μM)
at subsaturating cGMP (50 or 100 μM) at þ50 mV normalized to KD values determined at saturating cGMP (2 mM), versus 1 ꢀ I/Imax, which is related to
the fraction of closed channels. KD values at saturating cGMP were corrected for ion accumulation. Solid lines indicate simulations for exclusive closed
channel blockers, using KD/KDsat = (1 ꢀ Fsat)/(1 ꢀ FsatI/Imax), where Fsat is the estimated fraction of open heteromeric rod channels in saturating cGMP
assuming Fsat = 0.3 (a),49 0.56 (b),50 or 0.78 (c).51 The dotted line is a simulation for a blocker with no preferencefor state, or KD/KDsat = 1. The dashed line
ꢀ1
is a simulation for an exclusive open channel blocker, using KD/KDsat = (I/Imax
)
.
(Molecular Kinetics, Pullman, WA). Glass syringes and Teflon tubing
were used to minimize the binding of compounds to surfaces; concen-
trations passing through the perfusion system were verified by absor-
bance. Current traces were digitally filtered at 300 Hz (Gaussian) and
averaged using Clampfit 8.2 software. Currents in the absence of cGMP
were subtracted from all currents analyzed. KD values were determined
and expressed as the mean ( SD.
In Vitro Ester Hydrolysis. All enzymatic assays were performed
with 50 μM compound in 0.1 M phosphate buffer, pH 7.4, at 37 °C with
stirring, unless otherwise noted. Butyrylcholinesterase stock solutions
from human serum (Sigma, St. Louis, MO) were prepared at 100 U/mL
in 0.1 M phosphate buffer, pH 7.4, and stored at ꢀ20 °C until use.
Compound hydrolysis was monitored on an 8452A diode array spectro-
photometer (Hewlett-Packard). Product peak wavelength absorption
was immediately monitored upon addition of compound. Absorbances
for complete hydrolysis were determined when there were no further
detectable changes. Hydrolysis rates were calculated based on the
changes in absorbance during the first 1 min of hydrolysis (1 and 5)
or first 9 min (6 and 7) and were adjusted to the absorbance at the
completion of the reaction. All hydrolysis assays were performed in
triplicate, and final rate constants are expressed as the mean ( SD of the
three individually determined rate constants. In some experiments with
compound 1, samples were taken at the beginning and end of the assay
to verify the hydrolysis product and the completion of the reaction by
HPLC. These samples were compared to 1 and 3 on a C18 column
eluted with 0.1% TFA in water/acetonitrile.
Activity of chymotrypsin (bovine, Worthington Biochemical Co., 190
μg/mL) was verified with 90 μM 4-nitrophenyl acetate in 0.1 M HEPES
solution, pH 6.5, at 37 °C with stirring. Hydrolysis product was
monitored at 404 nm for 10 min. Activity of proteinase K (from
Engyodontium album, Sigma, 165 μg/mL) was verified with 16.5 mg/
mL bovine serum albumin in 0.1 M phosphate buffer, pH 7.4, at 37 °C.
Samples were taken at time point increments up to 2 h when the reaction
reached completion and were analyzed with 12% SDSꢀPAGE, stained
with 0.1% Coomassie Blue.
Statistical Analysis. Statistical comparison between groups was
made using a one-way repeated-measures ANOVA and the HolmꢀSidak
post hoc method for multiple pairwise comparisons. Statistical signifi-
cance was accepted at P < 0.01.
Chemistry. Reagents, including 1 and 2, were obtained from Sigma-
Aldrich and were used without further purification. TLC was performed
on glass backed silica plates and eluted in a mixture of 5ꢀ10% methanol
and 90ꢀ95% dichloromethane. Plates were visualized using short wave
UV light and KMnO4. Crude compounds were initially purified using
column chromatography, which was packed with normal phase silica gel
and eluted using either ethyl acetate/hexane or methanol/dichloro-
methane mixtures. Trace impurities were removed by reversed-phase
HPLC on an Xterra Prep RP8 column, 19 mm ꢂ 100 mm, 5 μm
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(Waters, Milford, MA), with a waterꢀmethanol gradient (5 mM
ammonium acetate, pH 5), monitored at 214 and 310 nm to yield final
products. Purity was assessed to be greater than 95% with an Xterra
Analytical RP8 column, 4.6 mm ꢂ 250 mm, 5 μm, under similar
4-(Octylamino)-N-(2-(dimethylamino)ethyl)benzamide (7).
The product was prepared as described for 6 using compound 4 to yield 7
(87%) as pale brown oil. 1H NMR (500 MHz) (CDCl3): δ 7.66 (d, J = 8.8
Hz, 2H), 6.99 (br, 1H), 6.55 (d, J = 8.8 Hz, 2H), 3.98 (br, 1H), 3.57
(m, 2H), 3.13 (t, J = 7.1 Hz, 2H), 2.68 (t, J = 5.7 Hz, 2H), 2.40 (s, 6H),
1.61 (m, 2H), 1.25ꢀ1.40 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR
(125 MHz) (CDCl3): δ 167.8, 151.4, 129.1, 122.4, 111.8, 58.4, 51.1, 45.2,
43.8, 36.9, 32.1, 29.7, 29.6, 27.4, 23.0, 14.4. ESI-MS: m/z 320.08 MHþ.
4-(Butylamino)-N-(2-(dimethylamino)ethyl)benzothioamide
(8). In a flame-sealed flask, 6 (∼0.25 mmol) was dissolved in 5 mL of dry
toluene with Lawesson’s reagent (1 mol equiv) and refluxed under argon for
2.5 h. The mixture was dissolved in 20 mL of ethyl acetate, washed with water
(2ꢂ 20 mL), dried over sodium sulfate, and subsequently purified via column
chromatography with 10% methanol in dichloromethane to yield 8 (26%) as
yellow oil. 1HNMR(500MHz) (CDCl3):δ8.64 (br, 1H), 7.79 (d, J=9Hz,
2H), 6.51 (d, J = 9 Hz, 2H), 4.03 (br, 1H), 3.97 (m, 2H), 3.14 (t, J = 7.1 Hz,
2H), 2.85 (m, 2H), 2.44 (s, 6H), 1.60 (m, 2H), 1.42 (m, 2H), 0.96 (t, J = 7.4
Hz, 3H). 13C NMR (125 MHz) (CDCl3): δ 197.6, 151.7, 129.3, 129.2,
111.7, 57.0, 45.1, 43.5, 43.2, 31.7, 20.5, 14.2. ESI-MS: m/z 280.1 MHþ.
4-(Octylamino)-N-(2-(dimethylamino)ethyl)benzothioamide
(9).The product was prepared as described for 8using compound 7to yield 9
(73%) as yellow oil. 1H NMR (500 MHz) (CDCl3): δ 8.41 (br, 1H), 7.76
(d,J= 8.8 Hz, 2H), 6.52 (d, J= 8.8 Hz, 2H), 4.02 (br, 1H), 3.90 (m, 2H), 3.14
(t, J = 7.1 Hz, 2H), 2.72 (t, J = 5.4 Hz, 2H), 2.35 (s, 6H), 1.62 (m, 2H),
1.25ꢀ1.40 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz)
(CDCl3): δ 197.4, 151.6, 129.4, 129.2, 111.8, 56.9, 51.2, 45.2, 43.8, 43.6, 32.2,
29.7, 29.6, 27.4, 23.0, 14.5. ESI-MS: m/z 336.13 MHþ.
1
conditions and monitoring. H and 13C NMR spectra were obtained
using a Bruker 500 MHz FT-NMR spectrometer. ESI-MS was per-
formed on a Thermo Finnigan TSQ Classic mass spectrometer.
4-(Butylamino)benzoic Acid (3). Compound 2 (∼3 mmol) was
dissolved in 15 mL of methanol with R-picoline-borane (1.1 mol equiv)
and butanal (1.1 mol equiv). The mixture was stoppered with a vent
needle and stirred overnight at room temperature. After 16ꢀ24 h,
solvent was removed in vacuo, 10 mL 1 M HCl was added to the flask,
and the mixture was stirred at room temperature for an additional 30
min. The pH was adjusted to neutral using NaHCO3, and the inter-
mediate product was extracted with ethyl acetate (2 ꢂ 60 mL). The
organic layer was washed with brine (1 ꢂ 45 mL), dried with magnesium
sulfate, filtered, removed in vacuo, and subsequently purified via column
chromatography with 30% ethyl acetate in hexane to yield 3 (88%) as a
white powder. 1H NMR (500 MHz) (CD3OD): δ 7.92 (d, J = 8.8 Hz,
2H), 6.55 (d, J = 8.8 Hz, 2H), 3.18 (t, J = 7.2 Hz, 2H), 1.63 (m, 2H), 1.44
(m, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz) (CD3OD):
δ 172.6, 153.1, 132.7, 117.3, 111.6, 43.4, 31.7, 20.5, 14.1.
4-(Octylamino)benzoic Acid (4). The product was prepared as
1
described for 3 with octanal to yield 4 (94%) as a white powder. H
NMR (500 MHz) (CD3OD): δ 7.92 (d, J = 8.9 Hz, 2H), 6.55 (d, J = 8.9
Hz, 2H), 3.17 (t, J = 7.2 Hz, 2H), 1.63 (m, 2H), 1.25ꢀ1.41 (m, 10H),
0.89 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz) (CD3OD): δ 172.6,
153.1, 132.7, 117.3, 111.6, 43.7, 32.1, 29.7, 29.6, 29.5, 27.4, 23.0, 14.4.
2-(Dimethylamino)ethyl 4-(Octylamino)benzoate (5). In a
flame-sealed flask, 4 (∼0.50 mmol) and CDI (1.5 mol equiv) were
dissolved in 3.0 mL of 1,2-dimethoxyethane (DME). The solution was
stirred at 60 °C for approximately 2 h under argon. 2-(Dimethylamino)-
ethanol (3 mol equiv) was added to the solution followed by a small
quantity of NaH (∼2 mg). The flask was allowed to cool to room
temperature and react for an additional 16ꢀ24 h. The reaction was
worked up by dissolving it in 100 mL of chloroform and washing with
water (2 ꢂ 60 mL) and brine (1 ꢂ 60 mL). The organic layer was dried
with sodium sulfate, filtered, removed in vacuo, and subsequently
purified via column chromatography with 30% ethyl acetate in hexane
to yield 5 (95%) as a white powder. This compound was reported
previously although synthesized by a different method.40 1H NMR (500
MHz) (CD3OD): δ 7.83 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 8.7 Hz, 2H),
4.58 (t, J = 5.0 Hz, 2H), 3.57 (t, J = 5.0 Hz, 2H), 3.14 (t, J = 7.1 Hz, 2H),
3.00 (s, 6H), 1.63 (m, 2H), 1.25ꢀ1.5 (m, 10 H), 0.90 (t, J = 6.8 Hz, 3H).
13C NMR (125 MHz) (CDCl3): δ 166.8, 152.2, 131.6, 117.9, 111.3,
62.3, 58.0, 45.9, 43.4, 31.8, 29.4, 29.3, 29.2, 27.1, 22.7, 14.1. ESI-MS: m/z
321.1 MH+. Mp: 40ꢀ41 °C.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 503-494-7463. Fax: 503-494-4352. E-mail: karpenj@oh-
su.edu.
’ ACKNOWLEDGMENT
This work was supported by National Institutes of Health
Grants EY009275 and MH071625 (J.W.K.) and Partners in
Science Program Grant 2009326 from the M. J. Murdock
Charitable Trust (S.R.K. and G.G.W.). We thank the Bioanaly-
tical Shared Resource at OHSU for mass spectrometry data,
Tapasree Banerji for technical assistance, and Michelle Schaffer
for helpful discussions.
’ ABBREVIATIONS USED
CDI, 1,10-carbonyldiimidazole; CNG, cyclic nucleotide-gated;
DME, 1,2-dimethoxyethane
4-(Butylamino)-N-(2-(dimethylamino)ethyl)benzamide (6).
In a flame-sealed flask, 3 (∼0.50 mmol) and CDI (1.5 mol equiv) were
dissolved in 3.0 mL of DME, and the mixture was stirred at 60 °C for
approximately 2 h under argon. N0,N0-Dimethylethane-1,2-diamine
(3 mol equiv) was added to the solution followed by a small quantity of
NaH (∼2 mg). The flask was allowed to cool to room temperature and
react for anadditional 16ꢀ24 h. The reaction was workedup by dissolving
itin100 mL ofchloroform and washing withwater (2 ꢂ 60 mL) and brine
(1 ꢂ 60 mL). The organic layer was dried over sodium sulfate, filtered,
removed in vacuo, and subsequently purified via column chromatography
with 30% ethyl acetate in hexane to yield 6 (93%) as pale brown oil. 1H
NMR (500 MHz) (CD3OD): δ 8.13 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8
Hz, 2H), 3.82 (t, J = 5.9 Hz, 2H), 3.44 (m, 4H), 3.02 (s, 6H), 1.76 (m,
2H), 1.50 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz)
(CD3OD): δ 170.2, 141.8, 135.8, 131.7, 124.0, 59.5, 53.1, 44.8, 37.3, 30.3,
21.6, 14.8. ESI-MS: m/z 264.22 MHþ.
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